| Description | Lactate dehydrogenase (LDH or LD) is a stable protein present in the cytoplasm of normal cells and normally cannot pass through the cell membrane. When cells are damaged, membrane permeability increases, and LDH is released extracellularly. A decrease in intracellular LDH and an increase in LDH in Lactate dehydrogenase (LDH or LD) is a stable protein present in the cytoplasm of normal cells and normally cannot pass through the cell membrane. When cells are damaged, membrane permeability increases, and LDH is released extracellularly. A decrease in intracellular LDH and an increase in LDH in the culture medium occur. Measuring the LDH activity in the culture medium or the LDH leakage rate can reflect drug-induced cytotoxicity. LDH belongs to the oxidoreductase family and can reversibly catalyze the redox reaction between lactate (L) and pyruvate (P). The reaction formula is: Lactate + NAD⁺ → Pyruvate + NADH + H⁺, where L → P is the forward reaction and P → L is the reverse reaction. Detection Principle: Using NAD⁺ as a hydrogen acceptor, LDH catalyzes the dehydrogenation of lactate to generate pyruvate. Pyruvate then reacts with dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which appears brownish-red in an alkaline solution. The color intensity is proportional to the pyruvate concentration. The absorbance at 440 nm can be measured using a microplate reader. The released LDH activity during cytotoxicity or the LDH activity in other samples can be calculated using formulas. This kit can be used for routine LDH activity detection and is more commonly used for cytotoxicity assays using LDH release as an indicator.This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes.L1501786Component100T500TStorageL1501786ALDH Assay Buffer3 mL15 mL2-8℃. Store in the dark.L1501786BNAD1EA2EA-20℃L1501786CPhenylhydrazine Color Solution3 mL15 mL2-8℃. Store in the dark.L1501786DAlkaline Color Solution10 mL50 mLRT.L1501786ELDH Releasing Agent (10X)2 mL10 mLRT.User-Prepared Instruments and Reagents1. 96-well plate cultured test and control group cell samples, sterile PBS, culture medium, distilled water.2. Microplate centrifuge, 96-well plate or centrifuge, centrifuge tubes, incubator or water bath, microplate reader.Experimental Procedure1. Sample Preparation1.1 LDH Release AssaySeed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.Aspirate the culture medium, wash once with PBS, add fresh culture medium.Set up corresponding control groups according to experimental needs:Background Blank Control Well A: Culture medium without cells.Sample Control Well B: Control cells without drug treatment.Maximum Enzyme Activity Control Well C: Lysed samples from untreated cells.Drug-treated Sample Well D: Cells treated with the drug.Continue cultivation.Before detection, take out the cell culture plate. Add LDH Releasing Agent (10X) to the "Maximum Enzyme Activity Control Well C" at a volume equal to 10% of the original culture medium volume. Mix thoroughly by pipetting up and down several times. Continue cultivation for about 1 hour.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent LDH detection.1.2 Cytotoxicity and Cell Proliferation Assay for Intracellular Total LDHSeed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.Treat with different drugs and set up appropriate controls.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate the culture medium.Add 150 µL of LDH Releasing Agent diluted 10-fold with PBS. Shake the plate to mix thoroughly. Continue cultivation for about 1 hour.Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent cytotoxicity detection.1.3 Protein Concentration DeterminationAfter sample preparation, the protein concentration can be determined using a BCA Protein Assay Kit (Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit are recommended) to facilitate subsequent calculation of LDH content per unit protein weight in tissues or cells.2. Preparation of NAD SolutionTake one vial of NAD (powder) and dissolve it in 1.5 mL of deionized water.3. LDH Enzymatic ReactionAdd solutions sequentially according to the table below, taking care to avoid bubbles. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.Reagent (µL)Volume (µL)Test Sample (supernatant)5LDH Assay Buffer25NAD Solution5 Mix well, incubate at 37°C for 15 min. Phenylhydrazine Color Solution25 Mix well, incubate at 37°C for 15 min. Alkaline Color Solution100Distilled Water150 4. LDH Measurement Mix well and let stand at room temperature for 5 minutes. Measure the absorbance of each well at 440 nm using a microplate reader. 5. Result Calculation Cytotoxicity or Mortality Rate (%) = (A D - A B ) / (A C - A B ) × 100% If the absorbance value A γ of a known concentration *c* of an LDH enzyme standard and the absorbance value A γ0 of the standard blank control are measured simultaneously, the enzyme activity in the sample can be roughly calculated:LDH Activity in Test Sample (mU/mL) = (A B - A A ) / (A γ - A γ0 ) × *c* For accurate calculation of the absolute LDH enzyme activity in the sample, use a self-prepared LDH standard to plot a standard curve with the measured absorbance values. The enzyme activity of the sample can be calculated using the formula derived from the standard curve. Where: A A = Absorbance of Background Blank Control Well A A B = Absorbance of Sample Control Well B A C = Absorbance of Maximum Enzyme Activity Control Well C A D = Absorbance of Drug-treated Sample Well D 6. Results and Analysis The cytotoxicity of drugs or toxicants can be determined by directly comparing the LDH activity in each well. Higher LDH activity indicates higher cell membrane permeability and more severe cell damage.Precautions1. Use serum-free or low-serum concentration culture medium when culturing cells to exclude serum interference; otherwise, deviations may occur.2. EDTA inhibits LDH. Avoid using or thoroughly remove reagents containing EDTA during operation.3. Measure LDH as soon as possible after collection. If the collected cell culture medium is stored for too long, LDH activity may decrease.4. Use solutions prepared at the same time for the same batch of experiments. The volume of solutions used and the reaction time should be consistent.5. In the enzymatic reaction, the recommended supernatant sample volume is 2.5-10 µL. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.6. Measurement should be completed within 15 minutes after color development.7. The Alkaline Color Solution is somewhat corrosive; handle with care.8. Use reagents promptly after opening to avoid affecting subsequent experimental results.9. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G Proteinase K 1.25 mL 4×1.25 mL RT B665530H Spin Columns DM with Collection Tubes 50 sets 200 sets RTProduct IntroductionThis reagent kit is suitable for extracting total DNA, including genomic DNA, mitochondrial DNA, and viral DNA, from fresh or frozen whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin), plasma, serum, erythrocyte sedimentation rate brown layer, lymphocytes, cell-free body fluids, and other samples. This product can process 0.1-1 mL of whole blood with a maximum yield of 30% µ g. It can purify DNA with sizes ranging from 100 bp to 50 kb. The purified DNA has high yield and good quality, and can remove protein, pigment, lipid, and other inhibitory impurities to the maximum extent. It can be directly used for PCR, fluorescence quantitative PCR, enzyme digestion, and Southern Blot experiments.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. The sample should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2. This reagent kit can extract up to 0.1-1 mL of whole blood samples or 1 × 107 white blood cells.3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in the Buffer GL. If there is any crystallization or precipitation, please incubate the Buffer GL in a 56 ℃ water bath and dissolve it again.5. The Buffer RCL in the reagent kit cannot be used again after being turbid.Operation steps:1. Sample processing: 1a When extracting 200 uL of blood sample, add the sample to the centrifuge tube (provided) and proceed directly to the next step of the experiment. 1b When the blood sample size is less than 200 µ When L, add Buffer GR to make up for 200 µ L. Proceed to the next step of the experiment. 1c When the blood sample size exceeds 200 µ When L is reached, add 1-2 times the volume of Buffer RCL, gently vortex or invert and mix well. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and carefully discard the supernatant. If there is still red in the sediment, repeat the above steps once. Then add 200 to the precipitate µ Shake the buffer GR until thoroughly mixed before proceeding to the next step of the experiment. 1d If the processed blood sample is anticoagulant from poultry, birds, amphibians, or lower level organisms, its red blood cells are nucleated cells, and the blood sample size is 5-20 µ L. Can be added to Buffer GR to make up to 200 µ Follow up experiments will be conducted afterwards. Note: If downstream experiments are sensitive to RNA, 4 can be added µ L RNase A (100mg/mL) solution, shake for 15 seconds, and leave at room temperature for 5 minutes. RNase A reagent kit is not provided. If needed, you can order it separately from our company, item number: CW0601S.2. Add 20 to the above solution µ L Protein K, mix well.3. Add 200 µ Shake with L Buffer GL until thoroughly mixed. Note: Do not pre mix Protein K and Buffer GL.4.Incubate at 4.56 ℃ for 10 minutes, invert and mix several times during this time. Attention: The DNA production has reached its maximum after 10 minutes of incubation, and further extension of incubation time has no effect on DNA production and purity.5. Add 200 µ L anhydrous ethanol, invert and mix several times. Short centrifugation causes the liquid on the tube wall and wall cover to concentrate at the bottom of the tube.6. Add all the solution obtained in step 5 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: If the extracted sample is the blood genome of species such as mice or monkeys that are difficult to remove heme, it is recommended to repeat step 7.8. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: To further improve DNA purity, repeat step 8.9.Centrifuge at 9.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.)10. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) If the final concentration of DNA needs to be increased, the obtained DNA eluent can be added back to the adsorption membrane, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute. 3) Because DNA stored in water is affected by acidic hydrolysis, if long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃... Read More | Inquire | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle. * This kit is suitable for human genomic DNA library construction, the starting template DNA input amount is 1 ng, our company also has 50 ng and 5 ng of human genomic DNA starting transposase method library construction kit, in order to get a higher quality library, different starting amount of DNA is recommended to use different kits. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for use with 1 ng of starting template DNA, and all reagents in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction.Product Features● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification preference.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kit: It is recommended to use Kangwei transposase method for second generation sequencing multi-sample primer kit.4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes.Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing. Sample PreparationDNA purity requirement: A260/A280 = 1.8-2.0. Sample DNA: dissolved in ultrapure water.DNA quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification). Schematic diagram of DNA banking process procedureDNA fragmentation, junction reaction 1. Add the following reagents to a 200 µl PCR tube:2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:inactivation reactionAfter the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.1. Add 3 µl of TS Buffer to the PCR tube containing the fragmentation product.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.PCR amplification1. Add the following reagents to a 200 µl PCR tube.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube. 3. Place the above PCR tubes in the PCR instrument with the thermal cap open, and the reaction program is as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads used is different, please refer to the attached table for the specific amount of magnetic beads used.(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 µl, add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds, and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Kangwei Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library.Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More |